Luminescence is the emission of light from excited electronic states of luminescent atoms or molecules (i.e., "luminophores"). Luminescence generally refers to all emission of light, except incandescence, and may include photoluminescence, chemiluminescence, and electrochemiluminescence, among others. In photoluminescence, which includes fluorescence and phosphorescence, the excited electronic state is created by the absorption of electromagnetic radiation. In particular, the excited electronic state is created by the absorption of radiation having an energy sufficient to excite an electron from a low-energy ground state into a higher-energy excited state. The energy associated with the excited state subsequently may be lost through one or more of several mechanisms, including production of a photon through fluorescence, phosphorescence, or other mechanisms. Here, the terms luminescence and photoluminescence are used interchangeably, except where noted, and a reference to luminescence or luminophore should be understood to imply a reference to photoluminescence and photoluminophore, respectively.
Luminescence may be characterized by a number of parameters, including luminescence lifetime. The luminescence lifetime is the average time that a luminophore spends in the excited state prior to returning to the ground state.
Luminescence may be used in assays to study the properties and environment of luminescent analytes. The analyte may be the focus of the assay, or the analyte may act as a reporter to provide information about another material or target substance that is the focus of the assay. Luminescence assays may be based on various aspects of the luminescence, including its intensity, polarization, and lifetime, among others. Luminescence assays also may be based on time-independent (steady-state) and/or time-dependent (time-resolved) properties of the luminescence.
Time-resolved luminescence assays may be used to study the temporal properties of a sample. These temporal properties generally include any properties describing the time evolution of the sample or components of the sample. These properties include the time-dependent luminescence emission and time-dependent luminescence polarization (or, equivalently, anisotropy), among others. These properties also include coefficients for describing such properties, such as the luminescence lifetime and the rotational (or more generally the reorientational) correlation time.
Time-resolved luminescence may be measured using "time-domain" or "frequency-domain" techniques, each of which involves monitoring the time course of luminescence emission.
In a time-domain measurement, the time course of luminescence is monitored directly. Typically, a sample containing a luminescent analyte is illuminated using a narrow pulse of light, and the time dependence of the intensity of the resulting luminescence emission is observed. For a simple luminophore, the luminescence commonly follows a single-exponential decay, so that the luminescence lifetime can (in principle) be determined from the time required for the intensity to fall to 1/e of its initial value.
In a frequency-domain measurement, the time course of luminescence is monitored indirectly, in frequency space. Typically, the sample is illuminated using intensity-modulated incident light, where the modulation may be characterized by a characteristic time, such as a period. Frequency-domain analysis may use almost any modulation profile. However, because virtually any modulation profile can be expressed as a sum of sinusoidal components using Fourier analysis, frequency-domain analysis may be understood by studying the relationship between excitation and emission for sinusoidal modulation.
FIG. 1 shows the relationship between excitation and emission in a frequency-domain experiment, where the excitation light is modulated sinusoidally at a single modulation frequency f. The resulting luminescence emission is modulated at the same frequency as the excitation light. However, the intensity of the emission will lag the intensity of the excitation by a phase angle (phase) .phi. and will be demodulated by a demodulation factor (modulation) M. Specifically, the phase .phi. is the phase difference between the excitation and emission, and the modulation M is the ratio of the AC amplitude to the DC offset for the emission, relative to the ratio of the AC amplitude to the DC offset for the excitation. The phase and modulation are related to the luminescence lifetime .tau. by the following equations: EQU .omega..tau.=tan(.phi.) (1) ##EQU1##
Here, .omega. is the angular modulation frequency, which equals 2.pi. times the modulation frequency. Significantly, unlike in time-domain measurements, the measured quantities (phase and modulation) re directly related to the luminescence lifetime. For maximum sensitivity, the angular modulation frequency should be roughly the inverse of the luminescence lifetime. Typical luminescence lifetimes vary from less than about 1 nanosecond to greater than about 10 milliseconds. Therefore, instruments for measuring luminescence lifetimes should be able to cover modulation frequencies from less than about 20 Hz to greater than about 200 MHz.
A similar approach may be used to study other temporal properties of a luminescent sample, such as time-resolved luminescence polarization, which may be characterized by a rotational (or more generally a reorientational) correlation time. The use of standard frequency-domain techniques to study such properties is described in the above-identified patent applications and in Joseph R. Lakowicz, Principles of Fluorescence Spectroscopy (2.sup.nd ed. 1999), each of which is incorporated herein by reference.
Frequency-domain measurements typically are conducted at high frequencies, especially for short-lifetime luminophores. To simplify these measurements, the emission signal may be converted to a lower frequency, as follows. In radio-frequency (RF) signal detection, an input frequency may be converted (heterodyned) to a fixed intermediate frequency (IF) by mixing it with (i.e., multiplying it by) a signal from a local oscillator (LO) of appropriate frequency. Multiplying two frequencies creates an output containing the sum and difference frequencies. One of these outputs is selected as the IF signal by filtering. The IF signal contains the phase and amplitude information of the original RF signal but at a more convenient (i.e., usually lower) fixed frequency. In frequency-domain heterodyne fluorometry, the RF emission signal is mixed with a second, coherent frequency, and the IF is the isolated difference frequency output. Typically, a gain-modulated detector performs the mixing step.
If the source and detector frequencies are the same in a heterodyning scheme, the method is called homodyning. Homodyning, by definition, results in a zero-frequency (DC) IF signal. The intensity is proportional to the cosine of the difference of the phase between the detector and the emission. To acquire the entire phase and modulation information of the emission signal, the phase difference may be stepped systematically between the source and detector modulation signals. Alternatively, the RF signal may be demodulated using two LO signals whose phases are 90 degrees apart. The two resulting signals, the in-phase (I) and quadrature (Q) signals, are the Cartesian representations of the phase and modulation (cosine and sine components).
Homodyning is commonly used to collect phase-resolved data with a single frequency reference and a fixed phase difference. By properly choosing the phase of the detector, one can suppress or enhance certain lifetimes. A disadvantage of homodyning relative to heterodyning is that homodyning is more affected by DC offsets in the mixing and detection electronics.
The heterodyne frequency-domain method has two significant advantages over time-domain methods: (1) an enhanced excitation duty cycle, and (2) measurement of phase and modulation.
An enhanced excitation duty cycle may be advantageous because it implies that a near maximal amount of luminescence is being excited from the sample. (The excitation duty cycle is the fraction of time that the system is illuminated.) If the illumination is a pure sine wave, the excitation duty cycle can be as large as 50%. However, if the illumination is a narrow pulse, as in multiharmonic phase and modulation fluorometry, the excitation duty cycle will be much lower, comparable to that for time-domain methods.
Measurement of phase and modulation may be advantageous because these quantities may be relatively unaffected by the DC luminescence intensity of the system, or by fluctuations in light source intensity, drift of electronic offsets, and errors in sample concentration. Conversely, intensity measurements, such as those used in time-domain methods, may be strongly affected by these factors, so that they must be corrected by normalization and/or calibration.
Despite these advantages, the heterodyne frequency-domain method has two significant disadvantages, especially relative to time-domain methods: (1) a reduced detection duty cycle, and (2) a low sensitivity.
A reduced detection duty cycle is a significant disadvantage because it reduces the amount of luminescence that is detected. (The detection duty cycle is the fraction of time that the detector can process light.) Typically, the detector is internally gated or gain modulated for the heterodyning step because the detector cannot respond externally to the high-frequency luminescence emission signal. If the luminescence is a pure sine wave, the detected signal optimally will be gated off 50% of the time, either by gating the signal or gating the detector.
A low sensitivity is a significant disadvantage because it requires higher quantities of reagents and/or longer analysis times, if a sample may be analyzed at all. This low sensitivity reflects in part the cumulative effects of dark noise, which becomes an ever larger fraction of the signal as light levels are reduced.